Genomic and non-genomic mechanisms of androgens in the lower urinary tract smooth muscle of healthy and ovariectomized rats

Abstract

Hormonal decline in women lead to Lower Urinary Tract Symptoms (LUTS) such as voiding frequency, urgency, nocturia, and urinary incontinence that negatively impact the women´s quality of life and social interaction. Hormone replacement therapy with testosterone has been used in postmenopausal women to alleviate certain symptoms of androgen deficiency such as loss of libido and bone and muscle mass, as well as the impaired cognitive function. However, few studies have focused on the understanding s of androgen replacement in LUTS of postmenopausal women. A recent study by our group evaluated the effects of testosterone replacement in the voiding dysfunction of 4-month ovariectomized rats. We showed that ovariectomy causes in vivo voiding alterations (increases of urinary frequency, and of basal, threshold and post-void pressures), as well as in vitro bladder alterations (bladder hypocontractility and urethral hypercontractility), all of which are reversed by a supraphysiological dose of testosterone. These protective effects of testosterone were independent of estrogen synthesis, since the inhibition of testosterone aromatization by letrozole treatment remained unchanged. However, the mechanism(s) of action of androgens in the lower urinary smooth muscle tract (bladder and urethra) under physiological and pathological conditions (ovariectomy) remain unclear, and it is not known either if it is the result of Androgen Receptor (AR) activation by genomic and/or non-genomic mechanisms. Preliminary results in isolated bladders of healthy rats showed that pre-incubation with testosterone (100 nM, 30 min) promotes bladder relaxation (37%, p <0.05) and increased urethral contraction (45%, p <0.05), suggesting an effect that favors the urinary continence. In the bladder of ovariectomized rats, testosterone significantly reversed the bladder hypocontractility and urethral hypercontractility. In addition, the incubation of tissues with flutamide (selective and competitive intracellular AR antagonist) prevented the testosterone effects in the physiological, but not in the ovariectomy condition, suggesting that in the first condition testosterone exerts a genomic effect, whereas in the second the effects are non-genomic. Recent studies in the vascular system indicate that testosterone produces vasodilation by the release of Nitric Oxide (NO) from nitrergic fibers. The bladder is supplied by a rich nitrergic innervation, and that NO released (added to the ²2/²3-adrenergic activation by norepinephrine) exerts an important role mediating bladder relaxation during the storage phase. Therefore, our hypothesis is that in the physiological condition testosterone induces nitrergic NO release, leading to reduction of bladder contraction, promoting urinary continence. In the ovariectomy, where testosterone normalizes the bladder hypocontractility, the interpretation of our data is not yet clear, but may involve positive regulation of muscarinic receptors (M2/M3; bladder) and adrenergic ±1 (urethra) receptors. With the present project we propose to study the effects of testosterone (and its metabolite dihydrotestosterone) on the bladder and urethra smooth muscle of healthy and ovariectomized rats. We will investigate the gene expression, localization and downstream signaling of the Androgen Receptor (AR), as well as the modulation by this hormone of receptors that modulate urinary continence such as muscarinic (M2 and M3), adrenergic (±1 and ²2, ²3) and angiotensin II (AT1) receptor, and the NO signaling pathway (nNOS- soluble guanylate cyclase (sGC) - GMPc). We will also evaluate the effects of androgen on the contractile and relaxing responses of the bladder and urethra in vitro, determining the contribution of voltage-independent and dependent calcium and potassium channels in the vesicourethral smooth muscle. (AU)